Construction Of Modified Live Virus Vaccine Of Equine Herpesvirus Type 1 And The Usage Of Ehv-1 As Virus Vector

Equine Herpesvirus 1 (EHV-1) is the causative agent of respiratory disease, abortions and neurological disorders in horses. Sequence analysis as well as reverse genetic studies shown that a single amino acid variation in the EHV-1 DNA polymerase (D752/N752) determines the virus’neuropathogenic potential. Glycoprotein C (gC) of EHV-1 was known to play important roles in the early steps of infection and in release of virions and contribute to viral virulence. Here, a gC-negative EHV-1 strain RacLll mutant with a D752 to N752 mutation in the polymerase was constructed. A complementing rabbit kidney cell line designated RK13_Pol expressing the non-neurological form of the polymerase (N752) was generated. A Pol-negative RacLl 1 mutant was then constructed by inserting EYFP in lieu of the authentic polymerase gene via co-transfection of RacLl 1 DNA and a shuttle plasmid into the RK13_Pol cell line. After a homogenous Pol-negative virus population was obtained that was unable to grow on non-complementing cells, homologous recombination was used again to introduce the N752 Pol variant, which was amplified from non-neurologic EHV-1 strain NY03. To delete the gC open reading frame from the L11_D752N mutant, the EYFP gene flanked with LoxP sites was used as a positive selection marker. By expressing Cre upon co-transfection, EYFP was finally excised and a gC-negative, non-neurological RacLll mutant, termed L11_D752N AgC, was engineered. L11_D752N AgC was shown to replicate less effectively in vitro with virus titer that was 20-fold reduced when compared to wild type and gC revertant, and impaired in case of attachment to and release from RK13 cells. Interestingly, plaque sizes induced by L11_D752N AgC were increased and 10% larger than those of wild type. Using FACS analysis, it was demonstrated for the first time that, gC is responsible for the MHC-1 down-regulation by EHV-1. In vivo, infection with this modified virus did not cause apparent bodyweight loss and clinic signs. Importantly, L11_D752N AgC can only be recovered at day 2 post infection from murine lungs. Upon challenge with wild type virus, apparent protection by L11_D752N AgC was observed with virus titers that was more than 100-fold decreased in lung tissues.Equine herpesvirus type 1 (EHV-1), a member of the subfamily Alphaherpesviridae, infects horses worldwide. Althogh all EHV-1 strains are capable of causing abortions and respiratory disease, only a few sets of strains can induce neurological disease. It is known that a single amino acid variation ar position 752 in EHV-1 DNA polymerase (D752/N752) determines the virus’neuropathogenic potential. While the D to N752 mutation affects the function of polymerase, which is believed to be indispensable for virus growth, the effect of a deletion of residue 752 on virus growth and pathogenicity is not clear. Here, an EHV-1 strain RacLll mutant with a deletion of residue 752 of the viral polymerase was constructed using two-step (en passant) Red mutagenesis. The mutant virus was then repaired to code for D752 or N752, respectively, with the same mutagenesis strategy. The correct genotypes of the final mutant viruses were confirmed by sequencing and restriction fragment length polymorphisms (RFLPs) analysis. After transfection of the A752 clone into a rabbit kidney (RK13) cell line, mutant virus could be recovered and was shown to grow with kinetics that were indistinguishable from those of parental, wild-type RacLll. The results clearly show that the absence of the residue 752 in the essential enzyme is not required for virus growth in vitro. In addition, biochemial assays of the△752 mutant addressed that the mutant is more resistance to Aphidicolin, a Pol-targeting drug, than either D752 or N752 gene type, indicating that the deletion of residue 752 does alter the function of EHV-1 DNA polymerase. The effect of the single amino acid deletion on virus pathogenicity will be tested in a murine infection model and ultimately in horses in the futher.Bluetongue virus (BTV) can infect most species of domestic and wild ruminants causing substantial morbidity and mortality and, consequently, high economical losses. In 2006, an epizootic of BTV serotype 8 (BTV-8) in northern Europe started and has caused significant disease in cattle and sheep before comprehensive vaccination was introduced. Equine herpesvirus type 1 (EHV-1), a member of subfamily Alphaherpesviridae, has been shown to be an efficient live virus vector. Here, the construction and characterization of a recombinant vectored vaccine that expresses VP2 of BTV-8 using EHV-1 as the delivery vehicle is described. The VP2 gene was synthesized after codon optimization and cloned into a generic transfer plasmid. Two-step (en passant) Red mutagenesis was employed to manipulate a bacterial artificial chromosome (BAC) of EHV-1 vaccine strain RacH and generate the recombinant vaccine. To avoid any unwanted recombination, the HCMV promoter upstream of the egfp gene in mini-F sequence of pRacHrv was replaced with the EF-la promoter. Afterwards, the vp2 gene was inserted in lieu of ORF1 and ORF2 in the RacH genome. Southern blot and restriction fragment length polymorphism analyses (RFLPs) confirmed the correct insertion of the transgene. The recombinant virus rH-VP2 was finally achieved by restoring gp2 encoding gene71, and shown to grow with smaller plaque size but comparable growth kinetics when compared with parental virus rRacH. Stable expression of VP2 by the recombinant EHV-1 was demonstrated using indirect immunofluorescence assay and western blotting. Vaccination/challenge experiments in ruminants are planned.